1. Field of the Invention
The present invention is directed to a new type of immunoassay, which includes a sensitive technique for the quantitative detection of low concentrations of molecules of a particular type such as molecules in solution.
2. Description of the Prior Art
It is desirable in certain circumstances to measure very low concentrations of certain organic compounds. In medicine, for example, it is very useful to determine the concentration of a given kind of molecule, usually in solution, which either exists naturally in physiological fluids (e.g. blood or urine) or which has been introduced into the living system (e.g. drugs or contaminants). Because of the rapidly advancing state of understanding of the molecular basis of both the normal and diseased states of living systems, there is an increasing need for methods of detection which are quantitative, specific to the molecule of interest, highly sensitive and relatively simple to implement. Examples of molecules of interest in a medical and/or biological context include, but are not limited to, drugs, sex and adrenal hormones, biologically active peptides, circulating hormones and excreted antigens associated with tumors. In the case of drugs, for example, it is often the case that the safe and efficacious use of a particular drug requires that its concentration in the circulatory system be held to within relatively narrow bounds, referred to as the therapeutic range.
One broad approach used to detect the presence of a particular compound, referred to as the analyte, is the immunoassay, in which detection of a given molecular species, referred to generally as the ligand, is accomplished through the use of a second molecular species, often called the antiligand, or the receptor, which specifically binds to the first compound of interest. The presence of the ligand of interest is detected by measuring, or inferring, either directly or indirectly, the extent of binding of ligand to antiligand. The ligand may be either monoepitopic or polyepitopic and is generally defined to be any organic molecule for which there exists another molecule (i.e. the antiligand) which specifically binds to said ligand, owing to the recognition of some portion of said ligand. Examples of ligands include macromolecular antigens and haptens (e.g. drugs). The antiligand, or receptor, is usually an antibody, which either exists naturally or can be prepared artificially. The ligand and antiligand together form a homologous pair. Throughout the text the terms antigen and antibody, which represent typical examples, are used interchangeably with the terms ligand and antiligand, respectively, but such usage does not signify any loss of generality. In some cases, the antibody would be the ligand and the antigen the antiligand, if it was the presence of the antibody that was to be detected.
Implementation of a successful immunoassay requires a detectable signal which is related to the extent of antigen-antibody binding which occurs upon the reaction of the analyte with various assay reagents. Usually that signal is provided for by a label which is conjugated to either the ligand or the antiligand, depending on the mode of operation of the immunoassay. Any label which provides a stable, conveniently detectable signal is an acceptable candidate. Physical or chemical effects which produce detectable signals, and for which suitable labels exist, include radioactivity, fluorescence, chemiluminescence, phosphorescence and enzymatic activity, to name a few.
Broadly speaking, immunoassays fall into two general categories--heterogeneous and homogeneous. In heterogeneous assays, the purpose of the label is simply to establish the location of the molecule to which it is conjugated--i.e. to establish whether the labeled molecule is free in solution or is part of a bound complex. Heterogeneous assays generally function by explicitly separating bound antigen-antibody complexes from the remaining free antigen and/or antibody. A method which is frequently employed consists of attaching one of the members of the homologous pair to a solid surface by covalent binding, physical absorption, or some other means. When antigen-antibody binding occurs, the resulting bound complexes remain attached to this solid surface (composed of any suitably inert material such as plastic, paper, glass, metal, polymer gel, etc.), allowing for separation of free antigen and/or antibody in the surrounding solution by a wash step. A variation on this method consists of using small (typically 0.05 to 20 microns) suspendable particles to provide the solid surface onto which either antigen or antibody is immobilized. Separation is effected by centrifugation of the solution of sample, reagents and suspendable beads at an appropriate speed, resulting in selective sedimentation of the support particles together with the bound complexes.
Notwithstanding the successful application of heterogeneous assay procedures, it is generally desirable to eliminate separation steps, since the latter are time-consuming, labor-intensive and sometimes the source of errors in the signal measurement. Furthermore, the more complicated protocols associated with heterogeneous assays make them less suitable for automated instrumentation of the kind needed for large-scale clinical applications. Consequently, homogeneous assays are more desirable. In the homogeneous format, the signal obtained from the labeled ligand or antiligand is modified, or modulated, in some systematic, recognizable way when ligand-antiligand binding occurs. Consequently, separation of the labeled bound complexes from the free labeled molecules is no longer required.
There exist a number of ways in which immunoassays can be carried out. For clarity a heterogeneous format is assumed, although each approach can be utilized (with varying degrees of success) in a homogeneous format, given a suitable label which is modulated by the binding reaction.
In the competitive mode, the analyte, assumed to be antigen, is allowed to compete with a known concentration of labeled antigen (provided in reagent form in the assay kit) for binding to a limited number of antibody molecules which are attached to a solid matrix. Following an appropriate incubation period, the reacting solution is washed away, ideally leaving just labeled antigen-antibody complexes attached to the binding surface, thereby permitting the signal from the labels to be quantitated.
In another method, called the sandwich mode, the analyte, again assumed to be antigen, reacts with an excess of surface-immobilized antibody molecules. After a suitable incubation period, an excess of label-conjugated antibody is added to the system. After this reaction has gone to essential completion, a wash step removes unbound labeled antibody and other sources of contamination, permitting measurement of the signal produced by labels which are attached to antibody-antigen-antibody complexes.
In yet another approach, called the indirect mode, the analyte, this time assumed to consist of specific antibody, is allowed to bind to surface-immobilized antigen which is in excess. The binding surface is then washed and allowed to react with label-conjugated antibody. After a suitable incubation period the surface is washed again, removing free labeled antibody and permitting measurement of the signal due to labeled antibody. The resulting signal strength varies inversely with the concentration of the starting (unknown) antibody, since labeled antibody can bind only to those immobilized antigen molecules which have not already complexed to the analyte.
One of the most sensitive immunoassays developed thusfar is the radioimmunoassay (RIA), in which the label is a radionuclide, such as I.sup.125, conjugated to either member of the homologous (binding) pair. This assay, which is necessarily heterogeneous, has achieved extremely high sensitivities, extending down to the vicinity of 10.sup.-17 molar for certain analytes. The obvious advantage of radioactive labeling, and the reason for the extremely high sensitivity of RIA-type assays, is that there exists negligible natural background radioactivity in the samples to be analyzed. Also, RIA is relatively insensitive to variations in the overall chemical composition of the unknown sample solution. However, the radioactive reagents are expensive, possess relatively short shelf lives and require the use of sophisticated, expensive instrumentation as well as elaborate safety measures for both their use and disposal. Hence, there is an increasing motivation to develop non-isotopic assays.
Fluorescence provides a potentially attractive alternative to radioactivity as a suitable label for immunoassays. For example, fluorescein (usually in the form of fluorescein isothiocyanate, or "FITC") and a variety of other fluorescent dye molecules can be attached to most ligands and receptors without significantly impairing their binding properties. Fluorescent molecules have the property that they absorb light over a certain range of wavelengths and (after a delay ranging from 10.sup.-9 to 10.sup.-4 seconds) emit light over a range of longer wavelengths. Hence, through the use of a suitable light source, detector and optics, including excitation and emission filters, the fluorescence intensity originating from labeled molecules can be determined.
Several heterogeneous fluorescence-based immunoassays (FIA) have been developed, including the FIAX/StiQ.TM. method (IDT Corp., Santa Clara, CA.) and the Fluoromatic.TM. method (Bio-Rad Corp., Richmond, CA.). In the former case, antigen is immobilized on an absorbant surface consisting of a cellulose-like polymer mounted on the end of a portable "dipstick", which is manually inserted into sample, reagent and wash solutions and ultimately into the fluorescence measuring instrument. A competitive reaction utilizing FITC-labeled monospecific antibody is typically employed. In the Bio-Rad assay kit, the solid surface is replaced by suspendable polyacrylamide gel microbeads which carry covalently-bound specific antibody. A sandwich mode is typically employed, with centrifugal sedimentation, followed by resuspension, of the beads for separation and measurement. Photon-counting techniques can be used to extend the sensitivity of the fluorescence intensity measurement.
Use of an enzyme as a label has produced a variety of useful enzyme immunoassays (EIA), the most popular of which is known as ELISA. In the typical heterogeneous format a sandwich-type reaction is employed, in which the ligand of interest, assumed here to be antigen, binds to surface-immobilized specific antibody and then to an enzyme-antibody conjugate. After suitable incubation, any remaining free enzyme conjugate is eliminated by a wash or centrifugation step. A suitable substrate for the enzyme is then brought into contact with the surface containing the bound complexes. The enzyme-substrate pair is chosen to provide a reaction product which yields a readily detectable signal, such as a color change or a fluorescence emission. The use of an enzyme as a label services to effectively amplify the contribution of a single labeled bound complex to the measured signal, because many substrate molecules can be converted by a single enzyme molecule.
As discussed previously, it is generally desirable to eliminate the separation steps associated with typical heterogeneous assays and, instead, use homogeneous techniques. One of the first homogeneous assays to be developed was the fluorescence polarization immunoassay. Here, the polarization of the emission of the fluorescent dye label is modulated to an extent which depends on the rate of rotational diffusion, or tumbling, of the label in solution. Free labeled molecules which rotate rapidly relative to the lifetime of their excited states emit light of relatively random polarization (assuming a linearly polarized exciting beam, for example). However, when the label becomes attached to a relatively large bound complex, the rate of tumbling becomes relatively slow, resulting in fluorescence emission of substantially linear polarization (i.e. essentially unchanged). Unfortunately, this technique is limited in practice to the detection of low molecular weight ligands, e.g. drugs, whose rate of tumbling is sufficiently rapid to produce a measurable change in fluorescence polarization upon binding to the antiligand. The extent of modulation of the signal, in any case, is quite small.
Another useful fluorescence-based homogeneous technique is the fluorescence excitation transfer immunoassay (FETI), also known simply as fluorescence quenching. Here, two different dye labels, termed the donor and the acceptor, or quencher are used. The pair has the property that when the labels are brought close together, i.e. to within distances characteristic of the dimensions of antigen-antibody complexes, there is non-radiative energy transfer between the electronically excited donor molecule and the acceptor. That is, the acceptor quenches the fluorescence emission of the donor, resulting in a decreased intensity of the latter. In a typical competitive mode, the donor label is attached to the ligand of interest and the acceptor label fixed to the specific antibody. When ligand is present in the unknown sample, some fraction of the acceptor-labeled antibody binds to the free ligand, leaving a fraction of the labeled ligand unquenched and therefore able to emit fluorescence radiation. The intensity of the latter increases with increasing analyte concentration.
The principal drawback of the FETI technique is the requirement that the donor-labeled ligand be relatively pure. Substantial concentrations of labeled impurities produce a large background signal, making detection of a small change due to complexing all the more difficult. Along these lines, U.S. Pat. No. 4,261,968 describes an assay in which the quantum efficiency of a fluorescent label is decreased when the labeled antigen becomes bound to the antibody, resulting in a decrease in the total fluorescence emission of the sample solution.
One of the main factors which limits the sensitivity and reproducibility of all non-isotopic assays to varying degrees is the presence of background false signals. For example, in fluorescence-based assays the use of untreated blood serum may yield relatively high and variable background fluorescence levels due to the presence of proteins, bilirubin and drugs. In addition, there may exist variations in the absolute fluorescence intensity from one sample to the next due to fluorescence from sample cell surfaces, light scattering from impurities in solution, aberrations on optical surfaces, temperature dependent effects, etc. Problems related to impurities are particularly troublesome in homogeneous assays. However, the background false signal contributions are often relatively constant in time for any given sample measurement. Hence, a very useful technique for reducing the background contribution without the necessity of making additional control measurements is to determine the time rate of change of the signal. Such a rate determination in the early stages of the antigen-antibody binding reaction (i.e. when the rate is largest) should, in principle, be independent of the (constant) background level.
In principle, then, the rate determining procedure can be applied to any homogeneous assay technique, with the added advantage that the binding reaction need not be taken to essential completion, thereby resulting in a faster assay measurement. However, this approach becomes less feasible or advantageous the smaller the total signal change due to binding, relative to the background level. Hence, there are invariably practical limitations to the sensitivity which can be achieved using any of the existing homogeneous non-isotopic immunoassays, given the typical courses of background false signals, interferences and nonspecific effects.